The long-term objective of the research is to uncover the biological function of ergothioneine, a thiolimidazole betaine which occurs widespread in mammalian tissues and in plants and microorganisms, with the hope that this may lead to an appreciation of the relationship of varying levels in human tissues to normal and abnormal metabolic processes. More immediate aims will be to study its relationship to the biochemistry of fungi, which are able to synthesize ergothioneine and related compounds. A systematic study of ergothioneine biosynthesis in Neurospora has been pursued in our laboratories in the expectation that knowledge of the factors controlling synthesis might help to establish function. We have recently completed the elucidation of the biosynthetic pathway, and our latest findings have shown that the last step involves sulfoxide formation between hercynine (histidine betaine) and cysteine, with the utilization of molecular oxygen. We wish to study the details of this unique enzymatic thiolation reaction, including the mechanism of formation of the sulfoxide and also its cleavage to ergothioneine by means of a pyridoxal-P enzyme. Knowledge of these mechanisms is of importance both as a contribution to basic enzymology and as an aid to understanding other thiolation reactions of biological interest. The finding that molecular oxygen and a sulfoxide are involved in the biosynthesis of ergothioneine may provide leads about the function of ergothioneine. It becomes important to establish whether the synthesis of ergothioneine is related to metabolic reactions concerned with the utilization of oxygen. We wish to study the possible relationship of ergothioneine to such enzymes as fungal catalase, peroxidase, and more particularly superxoide dismutasee which has been suggested to be a defense against oxygen toxicity in aerobic organisms. Again, because sulfoxides are very effective inhibitors of free radical chain reactions, we wish also to see whether the intermediate sulfoxide produced in ergothioneine biosynthesis has a biological function of this nature.